Fig 2: SLE patient T-bethi BDN cells rapidly differentiate in ASCs in the absence of BCR stimulation.(a–d) ASC generation from SLE BN cells requires early but transient BCR activation. Cartoon (a) depicting in vitro stimulation conditions to activate sort-purified T-betlo BN cells from SLE patients. BN cells were stimulated for 3 days with R848, cytokines (IFN?, IL-2, IL-21, BAFF) ± anti-Ig (Step 1) and then washed and recultured for an additional 3 days with the same stimuli ± anti-Ig (Step 2). Cells were analyzed by flow cytometry on day 6 (b–d). Phenotypic characterization (b) of IgDnegCD27neg BDN cells in cultures containing anti-Ig for all 6 days showing expression of T-bet, CD11c, FcRL5, CD21 and CXCR5 by the T-bethi BDN subset. The frequency (c) and number (d) of CD38hiCD27+ ASCs in cultures lacking anti-Ig (-,-), containing anti-Ig for all 6 days (+,+) or exposed to anti-Ig for the first 3 days only (+,-). (e) SLE patient T-bethi BDN cells rapidly differentiate in ASCs. Purified SLE B cell subsets (T-betlo BN, T-betlo CD11cnegCXCR5+ CD27negIgDneg DN1 memory cells, T-betlo CD27+ memory B cells (Bmem) and T-bethi CD11c+CXCR5neg DN2 cells) were stimulated with cytokines (IFN?, IL-21, IL-2, BAFF) and R848 for 2.5 days then counted and transferred to anti-IgG ELISPOT plates for 6 hr. The frequency of IgG ASCs derived from each B cell subset is shown. See Figure 8—figure supplement 1 for gating strategy to purify B cell subsets from SLE patients. Data shown in (c–d) are from a single SLE individual and are representative of 2 independent experiments. Data reported in (e) are representative of 3 independent experiments using B cells sorted from 3 different SLE donors. Statistical analyses were performed using one-way ANOVA with Tukey’s multiple comparison test (d–e). P values *<0.05, **<0.01, ***<0.001, ****<0.0001.

Fig 3: IL21R expression levels decline as B cells differentiate into ASCs.BN cells were activated for 3 days with anti-lg, R848, IFNY and IL-2, then washed and cultured for an additional 3 days with IL-21 and R848 (Be.Y2 cells). ASCs (CD27hiCD38hi and non-ASCs were identified in the Be.Y2 cultures (a) and IL21R expression levels were evaluated in each subset and are reported as histograms (b).

Fig 4: Metabolic inhibitor treatments impact Th1/Th2 polarization. CD4 T cells from healthy donors were treated with various inhibitors overnight on day 11 or 12 post-activation. Cells were restimulated with PMA/ionomycin for 4 h and subjected to intracellular cytokine staining for (A) IL-2, ***p < 0.0001 (B) IFN-?, *p = 0.0415 (C) IL-4, **p = 0.0089. (D) IL-21, **p = 0.0034 (E) IL-9, and (F) IL-17. The percentage of positive cells for each cytokine was quantified by flow cytometry and plotted for multiple donors. Statistical significance was determined by RM one-way ANOVA with Dunnett's multiple comparisons test. (G) CD4 T cells from healthy donors as in (A–F) were restimulated with PMA/ionomycin for 4 h and subjected to qRT-PCR. Expression data of each transcription factor was normalized to expression of the 18s rRNA housekeeping gene. All groups were then compared to DMSO-treated T cells that had not been restimulated to calculate fold changes. Statistical significance was determined by two-way ANOVA with Dunnett's multiple comparisons test. **p = 0.0066.

Fig 5: ASC development from BN precursors is enhanced in Th1 containing co-cultures.Cartoon (a) depicting day 6 paired co-cultures containing Th1 (Be1 co-cultures) or Th2 (Be2 co-cultures) effectors generated from the same HD, BN cells from a second allogeneic HD and exogenous IL-21 and IL-2. Flow cytometric analysis showing T-bet expression (b) on gated HD B cells (non-ASCs) from Be1 and Be2 co-cultures. Phenotyping (c) of day 6 B cell-gated Be1 cells showing T-bet expression in combination with other surface markers. (d–g) ASC development in HD day 6 paired Be1 and Be2 co-cultures showing representative flow plots (d) and frequencies (e) of CD38hiCD27+ ASCs in CD19+/lo-gated B lineage cells. Frequencies of IgM+ (f) or IgG+ (g) ASCs in day 6 paired Be1 and Be2 co-cultures. See Figure 2—figure supplement 1 for BN isolation strategy and characterization of polarized Th1 and Th2 effectors. See Figure 2—figure supplement 2 for gating strategy to identify IgG+ and IgM+ ASCs. See Figure 2—figure supplement 3 for proliferation analysis of B cells in paired day 6 HD Be1 and Be2 co-cultures. Analyses in (b–c) are from representative co-cultures (n > 30). Experiments (e–g) performed on 15 (e), 8 (f) or 6 (g) independent paired Be1 and Be2 co-cultures. Statistical analyses were performed using a non-parametric Wilcoxon paired t test (e) or paired Student’s t test (f–g). P values *<0.05, **<0.01, ****<0.0001.